Generally, when researching tiny regions of interest on a sample, researchers often employ a fluorescence microscope to observe the sample. The microscope may be a conventional wide-field, structured light or confocal microscope. The optical configuration of such a microscope typically includes a light source, illumination optics, objective lens, sample holder, imaging optics and a detector. Light emitted from the light source illuminates the region of interest on the sample after propagating through the illumination optics and the objective lens. Microscope objective forms a magnified image of the object that can be observed via eyepiece, or in case of a digital microscope, the magnified image is captured by the detector and sent to a computer for live observation, data storage, and further analysis.
In wide-field microscopes, the target is imaged using a conventional wide-field strategy as in any standard microscope, and collecting the fluorescence emission. Generally, the fluorescent-stained or labeled sample is illuminated with excitation light of the appropriate wavelength(s) and the emission light is used to obtain the image; optical filters and/or dichroic mirrors are used to separate the excitation and emission light.
Confocal microscopes utilize specialized optical systems for imaging. In the simplest system, a laser operating at the excitation wavelength of the relevant fluorophore is focused to a point on the sample; simultaneously, the fluorescent emission from this illumination point is imaged onto a small-area detector. Any light emitted from all other areas of the sample is rejected by a small pinhole located in front to the detector which transmits on that light which originates from the illumination spot. The excitation spot and detector are scanned across the sample in a raster pattern to form a complete image. There are a variety of strategies to improve and optimize speed and throughput which are well known to those skilled in this area of art.
Line confocal microscopes are a modification of the confocal microscope, wherein the fluorescence excitation source is a laser beam; however, the beam is focused onto a narrow line on the sample, rather than a single point. The fluorescence emission is then imaged on the optical detector through the slit which acts as the spatial filter. Light emitted from any other areas of the sample remains out-of-focus and as a result is blocked by the slit. To form a two-dimensional image the line is scanned across the sample while simultaneously reading the line camera. This system can be expanded to use several lasers and several cameras simultaneously by using an appropriate optical arrangement.
One type of line confocal microscope is disclosed in U.S. Pat. No. 7,335,898, which is incorporated by reference, wherein the optical detector is a two dimensional sensor element operated in a rolling line shutter mode whereby the mechanical slit can be omitted and the overall system design may be simplified. It is apparent, however, that confocal microscopy offers a partial solution to imaging 3D structures but does so at a significant cost of the ability to image dynamic structures. Most cells, especially in eukaryotes, are never exposed to intense light and consequently are poorly adapted to successfully withstand high photon fluxes. Current solutions for providing for effective live-cell imaging and thick or complex biological structures are limited and expensive. Deconvolving wide-field images can be effective for live-cell imaging but are limited for thick or complex imaging. Confocal microscopy is effective for thick or complex imaging but is limited in live-cell imaging capability.
Existing systems provide for confocality in one-dimension (Y) while delivering wide-field performance in the orthogonal direction (X). This may have the advantage of producing significant enhancements to contrast in the image while delivering superior live-cell performance over conventional point-scan or multi-point confocal systems. However, existing systems can do so at the cost of optical quality. Since the existing systems provide confocality in one dimension only, the optics in other dimensions are elongated yielding unappealing images. Applying conventional deconvolution to these images can improve resolution and contrast but fails to improve the asymmetry (elongation) caused by the asymmetric optics.